Complete Genome Sequence of Thermus aquaticus Y51MC23

RESEARCH ARTICLE Complete Genome Sequence of Thermus aquaticus Y51MC23 Phillip J. Brumm1*, Scott Monsma2, Brendan Keough2, Svetlana Jasinovica2, Erin Ferguson2, Thomas Schoenfeld2, Michael Lodes2, David A. Mead2 1 C5-6 Technologies LLC, Fitchburg, Wisconsin, United States of America, 2 Lucigen Corporation, Middleton, Wisconsin, United States of America * Abstract OPEN ACCESS Citation: Brumm PJ, Monsma S, Keough B, Jasinovica S, Ferguson E, Schoenfeld T, et al. (2015) Complete Genome Sequence of Thermus aquaticus Y51MC23. PLoS ONE 10(10): e0138674. doi:10.1371/journal.pone.0138674 Editor: Hodaka Fujii, Osaka University, JAPAN Received: April 22, 2015 Accepted: September 2, 2015 Published: October 14, 2015 Copyright: © 2015 Brumm et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All sequence files are available from the GenBank database (http://www. ncbi.nlm.nih.gov/genbank/) (Chr.sqn Chr, CP010822; Chr.sqn pTA14, CP010823; Chr.sqn pTA16, CP010824; Chr.sqn pTA69, CP010825; Chr.sqn pTA78, CP010826). Funding: This project was supported by an SBIR grant award (1R43HG007797-01A1) from the National Human Genome Research Institute. C5 6 Technologies LLC, and Lucigen Corporation provided support in the form of salaries for authors PB, SM, BK, SJ, EF, TS, ML & DM, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the Thermus aquaticus Y51MC23 was isolated from a boiling spring in the Lower Geyser Basin of Yellowstone National Park. Remarkably, this T. aquaticus strain is able to grow anaerobically and produces multiple morphological forms. Y51MC23 is a Gram-negative, rod-shaped organism that grows well between 50°C and 80°C with maximum growth rate at 65°C to 70°C. Growth studies suggest that Y51MC23 primarily scavenges protein from the environment, supported by the high number of secreted and intracellular proteases and peptidases as well as transporter systems for amino acids and peptides. The genome was assembled de novo using a 350 bp fragment library (paired end sequencing) and an 8 kb long span mate pair library. A closed and finished genome was obtained consisting of a single chromosome of 2.15 Mb and four plasmids of 11, 14, 70, and 79 kb. Unlike other Thermus species, functions usually found on megaplasmids were identified on the chromosome. The Y51MC23 genome contains two full and two partial prophage as well as numerous CRISPR loci. The high identity and synteny between Y51MC23 prophage 2 and that of Thermus sp. 2.9 is interesting, given the 8,800 km separation of the two hot springs from which they were isolated. The anaerobic lifestyle of Y51MC23 is complex, with multiple morphologies present in cultures. The use of fluorescence microscopy reveals new details about these unusual morphological features, including the presence of multiple types of large and small spheres, often forming a confluent layer of spheres. Many of the spheres appear to be formed not from cell envelope or outer membrane components as previously believed, but from a remodeled peptidoglycan cell wall. These complex morphological forms may serve multiple functions in the survival of the organism, including food and nucleic acid storage as well as colony attachment and organization. Introduction Thermus aquaticus YT-1 holds a special place in the history of microbiology. The thermophile was first isolated and cultured from a hot spring in Yellowstone National Park in 1969 [1]. The discovery of life at high temperatures was controversial at that time, but later shown to be quite PLOS ONE | DOI:10.1371/journal.pone.0138674 October 14, 2015 1 / 30 Thermus aquaticus Complete Genome Sequence manuscript. The specific roles of these authors are articulated in the 'author contributions' section. Competing Interests: Authors SM, BK, SJ, EF, TS, ML & DM were employees of Lucigen at the time of the study and own stock or stock options in Lucigen. PB was an employee of C5-6 Technologies, Inc at the time of the study and owned stock and stock options in the company. C5-6 Technologies, Inc was dissolved in December 2014 by the board of directors and no longer exists. No patents or products were developed from this work. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. prevalent as demonstrated by the isolation of Thermus strains from hot water heaters and other sources [2]. The subsequent discovery and characterization of Thermus aquaticus DNA polymerase resulted in the development of amplification and sequencing tools that have revolutionized nearly every field of biology and medicine [3]. Thermus species that have been isolated from hot springs around the globe include T. brockianus, T. thermophilus [3], T. oshimai [4], T. caliditerrae [5], T. arciformis [6], T. islandicus [7] T. igniterrae [8] and T. antranikianii [8]. Not all Thermus species have been found in hot springs. T. composti was isolated from an oyster mushroom compost [9] and T. scotoductus from a South African gold mine [10]. A number of isolates initially classified as Thermus species have been reclassified as Meiothermus species [11], based on phylogenetic and physiological differences such as lower optimum growth temperatures. A unique feature of Thermus aquaticus YT-1 is its unusual cellular morphology. Shortly after the initial T. aquaticus report, Brock described the presence of “rotund bodies”, which appeared to be an association of multiple cells connected by a combined outer envelope as visualized by electron microscopy [12]. Little is known about the prevalence or function of these rotund bodies; however, the only other organism reported to form them is Meiothermus ruber (formerly Thermus ruber) [13–15]. Because of the low levels of rotund bodies observed in cultures, it is unclear if their production is limited to T. aquaticus and a few other related species, or if it is a trait shared by all Thermus species. Micrographs demonstrating the remarkable morphological diversity of Thermus aquaticus Y51MC23 are presented here. There were 28 Thermus genome projects as of April 2015 (Genomes online database, https://gold.jgi-psf.org/index), 23 of which have retrievable sequence data. Complete genome sequences have been reported for T. thermophilus HB8 [16], T. thermophilus HB27[17], T. scotoductus SA-01 [10], T. oshimai JL-2 and T. thermophilus JL-18 [18, 19]. Most Thermus genomes have been left unfinished in permanent draft status (17/23), including Thermus sp. strain RL [20], Thermus sp. strain CCB_US3_UF1 [21], Thermus sp. 2.9 [22], T. thermophilus ATCC 33923 [23], and T. aquaticus Y51MC23 (http://www.ncbi.nlm.nih.gov/nuccore/ 218297404). The ability to close and finish microbial genomes (...truncated)